Most current HIV-1 vaccine candidates are replication-defective, administered intramuscularly, and unlikely to induce immune responses at mucosal surfaces. The goal of the proposed research is to develop an orally administered replication-competent reovirus-based HIV-1 vaccine that stimulates mucosal and systemic humoral immune responses against broadly neutralizing epitopes of HIV-1. Reovirus infects intestinal mucosa to stimulate potent immune responses, is naturally attenuated in humans, and can be manipulated to express vaccine antigens. These key features make reovirus an ideal HIV-1 vaccine vector. The new vaccine vector will harbor HIV-1 sequences encoding the membrane-proximal external region (MPER) of HIV-1 envelope glycoprotein gp41, which form a-helical coiled coils at the base of the gp41 trimer. Importantly, monoclonal antibodies (MAbs) directed against MPER neutralize infection by HIV-1 primary isolates in vitro and protect against HIV-1 infection in passively immunized non-human primates. The central hypothesis of this proposal is that MPER sequences inserted into a-helical regions of trimeric reovirus attachment protein s1 will retain native immunogenicity and elicit neutralizing antibody responses against HIV-1. Three specific aims are proposed to test the central hypothesis and develop reovirus-based HIV-1 vaccines. In Specific Aim 1, recombinant reoviruses will be generated that display MPER sequences in native trimeric conformation within surface-exposed regions of reovirus s1. MPER-expressing reoviruses will be tested for replication efficiency, stability during serial passage, and interactions with MPER-specific mAbs. A first-generation reovirus vector has been engineered in which MPER sequences incorporated into s1 retain the native MPER-specific mAb epitope, indicating that the proposed approach is feasible. In Specific Aim 2, recombinant reovirus vectors will be tested for induction of humoral immune responses after inoculation into rabbits. Sera from immunized rabbits will be tested for the presence of MPER-specific antibodies and the capacity to neutralize infection by HIV-1 Env-pseudotyped viruses and primary isolates. Second-generation vectors will be designed based on results of the initial immunogenicity studies. In Specific Aim 3, the reovirus vector that induces the most potent HIV-1 neutralizing humoral immune responses in rabbits will be tested for induction of humoral immune responses in rhesus macaques. Sera from immunized macaques will be tested for MPER-specific antibodies and HIV-1 neutralizing capacity. This research will lead to a better understanding of vaccine requirements for the induction of potent mucosal immune responses against HIV-1 and foster development of a new class of HIV-1 vaccines that target mucosal sites. PUBLIC HEALTH RELEVANCE: HIV-1 infection is a leading cause of infectious disease morbidity and mortality worldwide, and vaccines to prevent HIV-1 infection are not available. The proposed research will develop new orally administered reovirus-based HIV-1 vaccines that display neutralizing epitopes in native conformation. This work will advance knowledge about induction of mucosal immunity and establish the feasibility for challenge studies in non-human primates and immunogenicity trials in humans.